Phosphatidyhlcholine biosynthetic enzymes

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a phosphatidylcholine biosynthetic enzyme. The invention also relates to the construction of a chimeric gene encoding all or a portion of the phosphatidylcholine biosynthetic enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the phosphatidylcholine biosynthetic enzyme in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/155,626, filed Sep. 23, 1999.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingphosphatidylcholine biosynthetic enzymes in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol(PG) and diphosphatidylglycerol (DPG) are the major phospholipids foundin plant tissues. The distribution of these lipids among the variousorganelles of different tissues and among different plants has beencomprehensively studied. The pathways by which these lipids aresynthesized have also been studied extensively but very few of the plantenzymes involved in these pathways have been purified or theircorresponding genes cloned.

[0004] The precursor molecule common to the de novo synthesis of allphospholipids in prokaryotes and eukaryotes is phosphatidic acid (PA).Synthesis of PA occurs by the sequential acylation ofglycerol-3-phosphate by glycerol-3-phosphate acyltransferase and1-monoacylglycerol-3-phosphate acyltransferase, both of which utilizeacyl-CoA as a source of acyl moieties. PA may be converted toCDP-diacylglycerol by the action of the enzyme CDP-diacylglycerolsynthase (E.C. 2.7.7.41; also called CTP:phosphatidatecytidylyltransferase, phosphate cytidylyltransferase,phosphoethanolamine cytidylyltransferase, among others). This enzyme hasbeen characterized in yeast where it has been demonstrated to be highlyregulated (Homann et al. (1987) J. Bacteriol. 169:3276-3280). Whilephosphatidate cytidylyltransferase activity has been detected in thechloroplast, mitochondria and microsomes of several plants, no sequenceinformation of plant phosphatidate cytidylyltransferase has beenconfirmed. The sequence of an Arabidopsis thaliana putativephosphoethanolamine cytidylyltransferase was identified when thesequence of the chromosome 2 was determined (Lin et al. (1999) Nature402:761-768).

[0005] In castor bean endosperms PE is sequentially methylated to PC bymethyltransferases which utilize S-adenosylmethionine as the methyldonor. PE N-methyltransferase (EC 2.1.1.17) catalyzes the methylation ofPE to phosphatidyl methylethanolamine (PME) andphosphatidyl-N-methylethanolamine N-methyltransferase (EC 2.1.1.71; alsocalled phosphatidylethanolamine N-methyltransferase) catalyzes the twomethylations necessary to convert PME to PC (McGraw and Henry (1989)Genetics 122:317-330). The sequence of a plant phosphatidylethanolamineN-methyltransferase has yet to be determined.

[0006] Identification of the sequences encoding phosphoethanolaminecytidylyltransferase or phosphatidylethanolamine N-methyltransferase inplants will allow the manipulation of these genes in transgenic plants.

SUMMARY OF THE INVENTION

[0007] The present invention concerns an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) a first nucleotide sequence encoding a polypeptide of at least 40amino acids having at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 18, and 20, (b) a secondnucleotide sequence encoding a polypeptide of at least 200 amino acidshaving at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:12, 14, and 18, and (c) a third nucleotide sequence comprisingthe complement of the first or second nucleotide sequences.

[0008] In a second embodiment, it is preferred that the isolatedpolynucleotide of the claimed invention comprises nucleotide sequenceselected from: (a) a first nucleotide sequence of at least 100nucleotides having at least 80% identity based on the Clustal method ofalignment when compared to a nucleotide sequence selected from SEQ IDNOs:1, 3, 5, 7, 9, 17, and 19, and (b) a second nucleotide sequenceselected from the group consisting of SEQ ID NOs:11, 13, and 15.

[0009] In a third embodiment, this invention concerns an isolatedpolynucleotide comprising a nucleotide sequence of at least one of 60(preferably at least one of 40, most preferably at least one of 30)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19and the complement of such nucleotide sequences.

[0010] In a fourth embodiment, this invention relates to a chimeric genecomprising an isolated polynucleotide of the present invention operablylinked to at least one suitable regulatory sequence.

[0011] In a fifth embodiment, the present invention concerns an isolatedhost cell comprising a chimeric gene of the present invention or anisolated polynucleotide of the present invention. The host cell may beeukaryotic, such as a yeast or a plant cell, or prokaryotic, such as abacterial cell. The present invention also relates to a virus,preferably a baculovirus, comprising an isolated polynucleotide of thepresent invention or a chimeric gene of the present invention.

[0012] In a sixth embodiment, the invention also relates to a processfor producing an isolated host cell comprising a chimeric gene of thepresent invention or an isolated polynucleotide of the presentinvention, the process comprising either transforming or transfecting anisolated compatible host cell with a chimeric gene or isolatedpolynucleotide of the present invention.

[0013] In a seventh embodiment, the invention concerns aCTP:phosphoethanolamine cytidylyltransferase or aphosphatidylethanolamine N-methyltransferase polypeptide selected fromthe group consisting of (a) a first polypeptide of at least 40 aminoacids comprising at least 80% identity based on the Clustal method ofalignment compared to a polypeptide selected from SEQ ID NOs:2, 4, 6, 8,10, 18, and 20, and (b) a second polynucleotide of at least 200 aminoacids comprising at least 80% identity based on the Clustal method ofalignment compared to a polypeptide selected from the group consistingof SEQ ID NOs:12, 14, and 16.

[0014] In an eighth embodiment, the invention relates to a method ofselecting an isolated polynucleotide that affects the level ofexpression of a CTP:phosphoethanolamine cytidylyltransferase or aphosphatidylethanolamine N-methyltransferase polypeptide or enzymeactivity in a host cell, preferably a plant cell, the method comprisingthe steps of: (a) constructing an isolated polynucleotide of the presentinvention or an isolated chimeric gene of the present invention; (b)introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; (c) measuring the level of the CTP:phosphoethanolaminecytidylyltransferase or the phosphatidylethanolamine N-methyltransferasepolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide; and (d) comparing the level of theCTP:phosphoethanolamine cytidylyltransferase or thephosphatidylethanolamine N-methyltransferase polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide withthe level of the CTP:phosphoethanolamine cytidylyltransferase or thephosphatidylethanolamine N-methyltransferase polypeptide or enzymeactivity in the host cell that does not contain the isolatedpolynucleotide.

[0015] In a ninth embodiment, the invention concerns a method ofobtaining a nucleic acid fragment encoding a substantial portion of aCTP:phosphoethanolamine cytidylyltransferase or aphosphatidylethanolamine N-methyltransferase, preferably a plantCTP:phosphoethanolamine cytidylyltransferase or phosphatidylethanolamineN-methyltransferase, comprising the steps of: synthesizing anoligonucleotide primer comprising a nucleotide sequence of at least oneof 60 (preferably at least one of 40, most preferably at least one of30) contiguous nucleotides derived from a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,and 19, and the complement of such nucleotide sequences; and amplifyinga nucleic acid fragment (preferably a cDNA inserted in a cloning vector)using the oligonucleotide primer. The amplified nucleic acid fragmentpreferably will encode a substantial portion of aCTP:phosphoethanolamine cytidylyltransferase or aphosphatidylethanolamine N-methyltransferase amino acid sequence.

[0016] In a tenth embodiment, this invention relates to a method ofobtaining a nucleic acid fragment encoding all or a substantial portionof the amino acid sequence encoding a CTP:phosphoethanolaminecytidylyltransferase or a phosphatidylethanolamine N-methyltransferasepolypeptide comprising the steps of: probing a cDNA or genomic librarywith an isolated polynucleotide of the present invention; identifying aDNA clone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

[0017] In an eleventh embodiment, this invention concerns a composition,such as a hybridization mixture, comprising an isolated polynucleotideof the present invention.

[0018] In a twelfth embodiment, this invention concerns a method forpositive selection of a transformed cell comprising: (a) transforming ahost cell with the chimeric gene of the present invention or anexpression cassette of the present invention; and (b) growing thetransformed host cell, preferably a plant cell, such as a monocot or adicot, under conditions which allow expression of thephosphatidylcholine biosynthetic enzyme in an amount sufficient tocomplement an auxotroph to provide a positive selection means.

[0019] In a thirteenth embodiment, this invention relates to a method ofaltering the level of expression of a phosphatidylcholine biosyntheticenzyme in a host cell comprising: (a) transforming a host cell with achimeric gene of the present invention; and (b) growing the transformedhost cell under conditions that are suitable for expression of thechimeric gene wherein expression of the chimeric gene results inproduction of altered levels of the phosphatidylcholine biosyntheticenzyme in the transformed host cell.

[0020] A further embodiment of the instant invention is a method forevaluating at least one compound for its ability to inhibit the activityof a phosphatidylcholine biosynthetic enzyme, the method comprising thesteps of: (a) transforming a host cell with a chimeric gene comprising anucleic acid fragment encoding a phosphatidylcholine biosynthetic enzymepolypeptide, operably linked to suitable regulatory sequences; (b)growing the transformed host cell under conditions that are suitable forexpression of the chimeric gene wherein expression of the chimeric generesults in production of phosphatidylcholine biosynthetic enzyme in thetransformed host cell; (c) optionally purifying the phosphatidylcholinebiosynthetic enzyme expressed by the transformed host cell; (d) treatingthe phosphatidylcholine biosynthetic enzyme with a compound to betested; and (e) comparing the activity of the phosphatidylcholinebiosynthetic enzyme that has been treated with a test compound to theactivity of an untreated phosphatidylcholine biosynthetic enzyme, andselecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

[0021] The invention can be more fully understood from the followingdetailed description and the accompanying Sequence Listing which form apart of this application.

[0022] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 PhosphatidylcholineBiosynthetic Enzymes SEQ ID NO: (Amino Protein Clone Designation(Nucleotide) Acid) Corn phospho- cc71se-b.pk0008.g2 1 2 ethanolaminecytidylyltransferase Rice phospho- rls48.pk0009.h11 3 4 ethanolaminecytidylyltransferase Soybean phospho- sr1.pk0136.h8 5 6 ethanolaminecytidylyltransferase Wheat phospho- wle1n.pk0092.b3 7 8 ethanolaminecytidylyltransferase Wheat phosphatidyl- w1m96.pk028.h24 9 10ethanolamine N-methyltransferase Corn phospho- Contig of: 11 12ethanolamine p0121.cfrmp55r:fis cytidylyltransferase p0121.cfrmz88r Ricephospho- r1s48.pk0009.h11:fis 13 14 ethanolamine cytidylyltransferaseSoybean phospho- sr1.pk0136.h8:fis 15 16 ethanolaminecytidylyltransferase Wheat phospho- wle1n.pk0092.b3:fis 17 18ethanolamine cytidylyltransferase Wheat phosphatidyl-w1m96.pk028.h24:fis 19 20 ethanolamine N-methyltransferase

[0023] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In the context of this disclosure, a number of terms shall beutilized. The terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 60contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 30 contiguous nucleotidesderived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, or thecomplement of such sequences.

[0025] The term “isolated” polynucleotide refers to a polynucleotidethat is substantially free from other nucleic acid sequences, such asand not limited to other chromosomal and extrachromosomal DNA and RNA.Isolated polynucleotides may be purified from a host cell in which theynaturally occur. Conventional nucleic acid purification methods known toskilled artisans may be used to obtain isolated polynucleotides. Theterm also embraces recombinant polynucleotides and chemicallysynthesized polynucleotides.

[0026] The term “recombinant” means, for example, that a nucleic acidsequence is made by an artificial combination of two otherwise separatedsegments of sequence, e.g., by chemical synthesis or by the manipulationof isolated nucleic acids by genetic engineering techniques.

[0027] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0028] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof. The terms “substantially similar” and“corresponding substantially” are used interchangeably herein.

[0029] Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

[0030] For example, it is well known in the art that antisensesuppression and co-suppression of gene. expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by using nucleic acid fragments that do not share100% sequence identity with the gene to be suppressed. Moreover,alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not effectthe functional properties of the encoded polypeptide, are well known inthe art. Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue, such as glycine, or a more hydrophobic residue, such as valine,leucine, or isoleucine. Similarly, changes which result in substitutionof one negatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 60 (preferably at least one of40, most preferably at least one of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, and the complement of suchnucleotide sequences may be used in methods of selecting an isolatedpolynucleotide that affects the expression of a CTP:phosphoethanolaminecytidylyltransferase or a phosphatidylethanolamine N-methyltransferasein a host cell. A method of selecting an isolated polynucleotide thataffects the level of expression of a polypeptide in a virus or in a hostcell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial)may comprise the steps of: constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene of the presentinvention; introducing the isolated polynucleotide or the isolatedchimeric gene into a host cell; measuring the level of a polypeptide orenzyme activity in the host cell containing the isolated polynucleotide;and comparing the level of a polypeptide or enzyme activity in the hostcell containing the isolated polynucleotide with the level of apolypeptide or enzyme activity in a host cell that does not contain theisolated polynucleotide.

[0031] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6X SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2X SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65°C.

[0032] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Suitable nucleic acid fragments (isolated polynucleotidesof the present invention) encode polypeptides that are at least about70% identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein. Suitable nucleic acidfragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

[0033] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0034] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0035] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0036] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0037] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0038] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or may be composed of different elements derived from differentpromoters found in nature, or may even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. Promoters which cause a nucleic acidfragment to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro and Goldberg (1989)Biochemistry of Plants 15:1-82. It is further recognized that since inmost cases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity.

[0039] “Translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

[0040] “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

[0041] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0042] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single polynucleotide so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0043] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0044] A “protein” or “polypeptide” is a chain of amino acids arrangedin a specific order determined by the coding sequence in apolynucleotide encoding the polypeptide. Each protein or polypeptide hasa unique function.

[0045] “Altered levels” or “altered expression” refers to the productionof gene product(s) in transgenic organisms in amounts or proportionsthat differ from that of normal or non-transformed organisms.

[0046] “Auxotroph” refers here to an organism that requires a specificgrowth factor (an amino acid, for example) for its growth.

[0047] “Mature protein” or the term “mature” when used in describing aprotein refers to a post-translationally processed polypeptide; i.e.,one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor protein” or the term“precursor” when used in describing a protein refers to the primaryproduct of translation of mRNA; i.e., with pre- and propeptides stillpresent. Pre- and propeptides may be but are not limited tointracellular localization signals.

[0048] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

[0049] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

[0050] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0051] “PCR” or “polymerase chain reaction” is well known by thoseskilled in the art as a technique used for the amplification of specificDNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0052] The present invention concerns an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) a first nucleotide sequence encoding a polypeptide of at least 40amino acids having at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 18, and 20, (b) a secondnucleotide sequence encoding a polypeptide of at least 200 amino acidshaving at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:12, 14, and 18, and (c) a third nucleotide sequence comprisingthe complement of the first or second nucleotide sequences.

[0053] In a second embodiment, it is preferred that the isolatedpolynucleotide of the claimed invention comprises nucleotide sequenceselected from: (a) a first nucleotide sequence of at least 100nucleotides having at least 80% identity based on the Clustal method ofalignment when compared to a nucleotide sequence selected from SEQ IDNOs:1, 3, 5, 7, 9, 17, and 19, and (b) a second nucleotide sequenceselected from the group consisting of SEQ ID NOs:11, 13, and 15.

[0054] Nucleic acid fragments encoding at least a portion of severalphosphatidylcholine biosynthetic enzymes have been isolated andidentified by comparison of random plant cDNA sequences to publicdatabases containing nucleotide and protein sequences using the BLASTalgorithms well known to those skilled in the art. The nucleic acidfragments of the instant invention may be used to isolate cDNAs andgenes encoding homologous proteins from the same or other plant species.Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to, methods of nucleic acid hybridization, and methodsof DNA and RNA amplification as exemplified by various uses of nucleicacid amplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

[0055] For example, genes encoding other phosphoethanolaminecytidylyltransferases or phosphatidylethanolamine N-methyltransferases,either as cDNAs or genomic DNAs, could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, an entiresequence can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, end-labeling techniques, or RNA probes using available invitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

[0056] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165). Consequently, a polynucleotidecomprising a nucleotide sequence of at least one of 60 (preferably oneof at least 40, most preferably one of at least 30) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and thecomplement of such nucleotide sequences may be used in such methods toobtain a nucleic acid fragment encoding a substantial portion of anamino acid sequence of a polypeptide.

[0057] The present invention relates to a method of obtaining a nucleicacid fragment encoding a substantial portion of aCTP:phosphoethanolamine cytidylyltransferase or aphosphatidylethanolamine N-methyltransferase polypeptide, preferably asubstantial portion of a plant CTP:phosphoethanolaminecytidylyltransferase or phosphatidylethanolamine N-methyltransferase,comprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 60 (preferably atleast one of 40, most preferably at least one of 30) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, and thecomplement of such nucleotide sequences; and amplifying a nucleic acidfragment (preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a portion of a CTP:phosphoethanolamine cytidylyltransferaseor a phosphatidylethanolamine N-methyltransferase polypeptide.

[0058] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length CDNA clones of interest(Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0059] In another embodiment, this invention concerns viruses and hostcells comprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

[0060] The nucleic acid fragments of the instant invention may be usedto create transgenic plants in which the disclosed polypeptides arepresent at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the ratio ofphosphoethanolamine/phosphotidylcholine (PE/PC) in those cells. PC is atrimethylated form of PE; overexpression of phosphatidylethanolamineN-methyltransferase will lead to more PC from PE. It might be necessaryto increase PE production for this to work well. Phosphoethanolaminecytidylyltransferase controls the synthesis of PE so its overexpressionshould yield higher levels of PE. Lecithin is extracted from soy oilduring processing and is a mix of PE and PC. It is used as an emulsifierin chocolate (to replace cocoa butter) and as a nutritional supplement.Soybean lecithin is fractionated to obtain a higher content of PC to besold as a nutritional supplement. Increasing the PC content of soybeanlecithin from its normal 14% to 35% will improve its value asnutritional supplement. Increasing the PC content of soybean to 25% willimprove its emulsifying qualities and it would be possible to replacecocoa butter in chocolate, thereby increasing its commercial value.

[0061] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

[0062] Plasmid vectors comprising the instant isolated polynucleotide(or chimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

[0063] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

[0064] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0065] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0066] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppressiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds. Forexample, one can screen by looking for changes in gene expression byusing antibodies specific for the protein encoded by the gene beingsuppressed, or one could establish assays that specifically measureenzyme activity. A preferred method will be one which allows largenumbers of samples to be processed rapidly, since it will be expectedthat a large number of transformants will be negative for the desiredphenotype.

[0067] In another embodiment, the present invention concerns apolypeptide of at least 40 amino acids that has at least 80% identitybased on the Clustal method of alignment when compared to a polypeptideselected from SEQ ID NOs:2, 4, 6, 8, 10, 18, or 20; or a polypeptide ofat least 200 amino acids that has at least 80% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NOs:12, 14, and 16.

[0068] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded phosphatidylcholine biosynthetic enzyme. An example of avector for high level expression of the instant polypeptides in abacterial host is provided (Example 7).

[0069] Additionally, the instant polypeptides can be used as a target tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze various steps inphosphatidylcholine biosynthethesis. Accordingly, inhibition of theactivity of one or more of the enzymes described herein could lead toinhibition of plant growth. Thus, the instant polypeptides could beappropriate for new herbicide discovery and design.

[0070] All or a substantial portion of the polynucleotides of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and used asmarkers for traits linked to those genes. Such information may be usefulin plant breeding in order to develop lines with desired phenotypes. Forexample, the instant nucleic acid fragments may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Maniatis)of restriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

[0071] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4:37-41. Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0072] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0073] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeRes. 5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

[0074] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

EXAMPLES

[0075] The present invention is further defined in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

[0076] The disclosure of each reference set forth in here isincorporated herein by reference in its entirety.

EXAMPLE 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

[0077] cDNA libraries representing mRNAs from various corn, rice,soybean, and wheat tissues were prepared. The characteristics of thelibraries are described below. TABLE 2 cDNA Libraries from Corn, Rice,Soybean, and Wheat Library Tissue Clone cc71se-b Corn Callus Type IITissue, Somatic cc71sc-b.pk0008.g2 Embryo Formed p0121 Corn Shank EarTissue Collected 5 p0121.cfrmp55r Days After Pollination* p0121 CornShank Ear Tissue Collected p0121.cfrmz88r 5 Days After Pollination*r1s48 Rice Leaf 15 Days After Germination, r1s48.pk0009.h11 48 HoursAfter Infection of Strain Magaporthe grisea 4360-R-67 (AVR2-YAMO);Susceptible sr1 Soybean Root sr1.pk0136.h8 wle1n Wheat Leaf From 7 DayOld Etiolated wle1n.pk0092.b3 Seedling* w1m96 Wheat Seedlings 96 HoursAfter w1m96.pk028.h24 Inoculation With Erysiphe graminis f. sp tritici

[0078] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

[0079] Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

[0080] Confirmed templates are transposed via the Primer Islandtransposition kit (PE Applied Biosystems, Foster City, Calif.) which isbased upon the Saccharomyces cerevisiae Tyl transposable element (Devineand Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitrotransposition system places unique binding sites randomly throughout apopulation of large DNA molecules. The transposed DNA is then used totransform DH10B electro-competent cells (Gibco BRL/Life Technologies,Rockville, Md.) via electroporation. The transposable element containsan additional selectable marker (named DHFR; Fling and Richards (1983)Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agarplates of only those subclones containing the integrated transposon.Multiple subclones are randomly selected from each transpositionreaction, plasmid DNAs are prepared via alkaline lysis, and templatesare sequenced (ABI Prism dye-terminator ReadyReaction mix) outward fromthe transposition event site, utilizing unique primers specific to thebinding sites within the transposon.

[0081] Sequence data is collected (ABI Prism Collections) and assembledusing Phred/Phrap (P. Green, University of Washington, Seattle).Phrep/Phrap is a public domain software program which re-reads the ABIsequence data, re-calls the bases, assigns quality values, and writesthe base calls and quality values into editable output files. The Phrapsequence assembly program uses these quality values to increase theaccuracy of the assembled sequence contigs. Assemblies are viewed by theConsed sequence editor (D. Gordon, University of Washington, Seattle).

EXAMPLE 2 Identification of cDNA Clones

[0082] cDNA clones encoding phosphatidylcholine biosynthetic enzymeswere identified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

[0083] ESTs submitted for analysis are compared to the genbank databaseas described above. ESTs that contain sequences more 5- or 3-prime canbe found by using the BLASTn algorithm (Altschul et al (1997) NucleicAcids Res. 25:3389-3402.) against the Du Pont proprietary databasecomparing nucleotide sequences that share common or overlapping regionsof sequence homology. Where common or overlapping sequences existbetween two or more nucleic acid fragments, the sequences can beassembled into a single contiguous nucleotide sequence, thus extendingthe original fragment in either the 5 or 3 prime direction. Once themost 5-prime EST is identified, its complete sequence can be determinedby Full Insert Sequencing as described in Example 1. Homologous genesbelonging to different species can be found by comparing the amino acidsequence of a known gene (from either a proprietary source or a publicdatabase) against an EST database using the tBLASTn algorithm. ThetBLASTn algorithm searches an amino acid query against a nucleotidedatabase that is translated in all 6 reading frames. This search allowsfor differences in nucleotide codon usage between different species, andfor codon degeneracy.

EXAMPLE 3 Characterization of cDNA Clones Encoding CTPPhosphoethanolamine Cytidylyltransferase

[0084] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the contig toa putative phosphoethanolamine cytidylyltransferase from Arabidopsisthaliana (NCBI General Identifier No. 3786005) and by the cDNAs to CTPphosphoethanolamine cytidylyltransferase from Homo sapiens and Rattusnorvegicus (NCBI General Identifier Nos. 4505651 and 3396102,respectively). Shown in Table 3 are the BLAST results for individualESTs and the NCBI General Identifier Nos. of the two sequences withclosest homology: TABLE 3 BLAST Results for Sequences EncodingPolypeptides Homologous to Phosphoethanolamine CytidylyltransferaseClone NCBI General Identifier No. BLAST pLog Score cc71se-b.pk0008.g24505651 33.00 3786005 76.70 r1s48.pk0009.h11 4505651 17.00 3786005 34.30sr1.pk0136.h8 4505651 25.15 3786005 50.70 wle1n.pk0092.b3 3396102 8.303786005 26.52

[0085] The sequence of the entire cDNA insert in clonecc71se-b.pk0008.g2 could not be obtained, thus a corn contig wasidentified that covers most of the same region at the 5′ end and islonger at the 3′ terminus. The sequence of the entire cDNA insert in theremaining clones listed in Table 3 was determined. The sequences towardsthe 5′ terminus from the rice and soybean sequences were obtained usingPCR techniques well known to those skilled in the art.

[0086] The BLASTX search using the EST sequences from clones listed inTable 4 revealed similarity of the polypeptides encoded by the Contigsto putative phospholipid cytidylyltransferase from Arabidopsis thaliana(NCBI General Identifier No. 3786005) and CG5547 gene product [alt 2]from Drosophila melanogaster (NCBI General Identifier No. 7298016), andby the cDNAs to CTP:phosphoethanolamine cytidylyltransferase from Homosapiens and Rattus norvegicus (NCBI General Identifier Nos. 4505651 and3396102, respectively). Shown in Table 4 are the BLAST results for thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”), the sequences of contigs assembled from two or more ESTs(“Contig”), or sequences assembled from FIS and 5′ PCR encoding theentire protein (“CGS”): TABLE 4 BLAST Results for Sequences EncodingPolypeptides Homologous to CTP:Phosphoethanolamine CytidylyltransferaseBLAST pLog Score Clone Status 3786005 7298016 4505651 3396102 Contig of:Contig 97.52 62.00 57.22 56.40 p0121.cfrmp55r:fis p0121.cfrmz88rr1s48.pk0009.h11:fis CGS >180.00 85.52 90.70 87.40 sr1.pk0136.h8:fis CGS173.00 78.52 80.10 78.52 wle1n.pk0092.b3:fis FIS 24.52 4.15 7.00 18.52

[0087] The data in Table 5 presents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 8,12, 14, 16, and 18 and the Arabidopsis thaliana and Homo sapienssequences ((NCBI General Identifier Nos. 3786005 and 4505651). TABLE 5Percent Identity of Amino Acid Sequences Deduced From the NucleotideSequences of cDNA Clones Encoding Polypeptides Homologous toCTP:Phosphoethanolamine Cytidylyltransferase Percent Identity to SEQ IDNO. 3786005 4505651 2 79.6 40.8 4 71.1 33.7 6 89.8 50.9 8 77.2 40.4 1269.4 36.2 14 73.4 39.6 16 58.7 37.5 18 70.8 31.9

[0088] Nucleotides 846 through 1409 from corn clone having SEQ ID NO:11are 96% identical to nucleotides 563 through 1 from corn EST having NCBIGeneral Identifier No. 6012413. Nucleotides 1372 through 1827 from riceclone having SEQ ID NO:13 are 96% identical to nucleotides 1 through 443from rice EST having NCBI General Identifier No. 3760654. Nucleotides1422 through 1840 from soybean clone having SEQ ID NO:15 are 99%identical to nucleotides 1 through 519 from soybean EST having NCBIGeneral Identifier No. 7285973.

[0089] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of two cornCTP:phosphoethanolamine cytidylyltransferase variants, a substantialportion and the entire rice and soybean CTP:phosphoethanolaminecytidylyltransferases and substantial portions of one wheatCTP:phosphoethanolamine cytidylyltransferase. These sequences representthe first corn, rice, soybean, and wheat sequences encodingCTP:phosphoethanolamine cytidylyltransferase known to Applicant.

EXAMPLE 4 Characterization of cDNA Clones EncodingPhosphatidylethanolamine N-Methyltransferase

[0090] The BLASTX search using the EST sequences from clone listed inTable 6 revealed similarity of the polypeptides encoded by the cDNAs tophosphatidylethanolamine N-methyltransferase from Homo sapiens (NCBIGeneral Identifier No. 5459516). Shown in Table 6 are the BLAST resultsfor individual EST: TABLE 6 BLAST Results for Sequences EncodingPolypeptides Homologous to Phosphatidylethanolamine N-MethyltransferaseBLAST pLog Score Clone Status 5459516 w1m96.pk028.h24 EST 34.9

[0091] The sequence of the entire cDNA insert in clone listed in Table 6was determined. The BLASTX search using the EST sequences from clonelisted in Table 7 revealed similarity of the polypeptides encoded by thecDNAs to phosphatidylethanolamine N-methyltransferase from Saccharomycescerevisiae (NCBI General Identifier No. 6322533). Shown in Table 7 arethe BLAST results for the sequences of the entire cDNA insert comprisingthe indicated cDNA clone encoding the entire protein (“CGS”): TABLE 7BLAST Results for Sequences Encoding Polypeptides Homologous toPhosphatidylethanolamine N-Methyltransferase BLAST pLog Score CloneStatus 6322533 w1m96.pk028.h24:fis CGS 58.22

[0092] The data in Table 8 presents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:10 and 20and the Saccharomyces cerevisiae sequence (NCBI General Identifier No.6322533). TABLE 8 Percent Identity of Amino Acid Sequences Deduced Fromthe Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologousto Phosphatidylethanolamine N-Methyltransferase Percent Identity to SEQID NO. 6322533 10 53.5 20 54.4

[0093] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion and an entire wheatphosphatidylethanolamine N-methyltransferase. These sequences representthe first plant sequences encoding phosphatidylethanolamineN-methyltransferase known to Applicant.

EXAMPLE 5 Expression of Chimeric Genes in Monocot Cells

[0094] A chimeric gene comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega; Madison, Wis.).Vector and insert DNA can be ligated at 15° C. overnight, essentially asdescribed (Maniatis). The ligated DNA may then be used to transform E.coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptides, and the 10 kD zein 3′region.

[0095] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0096] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0097] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 □m in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0098] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0099] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0100] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

EXAMPLE 6 Expression of Chimeric Genes in Dicot Cells

[0101] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol Chem. 261:9228-9238) can be used for expression ofthe instant polypeptides in transformed soybean. The phaseolin cassetteincludes about 500 nucleotides upstream (5′) from the translationinitiation codon and about 1650 nucleotides downstream (3′) from thetranslation stop codon of phaseolin. Between the 5′ and 3′ regions arethe unique restriction endonuclease sites Nco I (which includes the ATGtranslation initiation codon), Sma I, Kpn I and Xba I. The entirecassette is flanked by Hind III sites.

[0102] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0103] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0104] Soybean embryogenic suspension cultures can be maintained in 35mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0105] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0106] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0107] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0108] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0109] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

EXAMPLE 7 Expression of Chimeric Genes in Microbial Cells

[0110] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0111] Plasmid DNA containing a CDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% low melting agarose gel. Buffer and agarosecontain 10 μg/ml ethidium bromide for visualization of the DNA fragment.The fragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0112] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol.Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25°. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

EXAMPLE 8 Evaluating Compounds for Their Ability to Inhibit the Activityof Phosphatidylcholine Biosynthetic Enzymes

[0113] The polypeptides described herein may be produced using anynumber of methods known to those skilled in the art. Such methodsinclude, but are not limited to, expression in bacteria as described inExample 6, or expression in eukaryotic cell culture, in planta, andusing viral expression systems in suitably infected organisms or celllines. The instant polypeptides may be expressed either as mature formsof the proteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

[0114] Purification of the instant polypeptides, if desired, may utilizeany number of separation technologies familiar to those skilled in theart of protein purification. Examples of such methods include, but arenot limited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

[0115] Crude, partially purified or purified enzyme, either alone or asa fusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. For example, assays for phosphoethanolaminecytidylyltransferase are presented by Sundler (1975) J. Biol. Chem.250:8585-8590. Assays for phosphatidylethanolamine N-methyltransferaseare presented by Vance and Schneider (1981) Methods Enzymol. 71:581-588.

1 20 1 530 DNA Zea mays 1 cagcgacgac gagatcaagg ccaacaaggg accccccgtcacgccgctcc acgagagaat 60 ggtaatggtc cgtgctgtga aatgggtgga tgatatcattccagatgcac cttatgccat 120 aactgaagaa ttcatgaaca aactattcaa tgagtacaacatagactaca ttatccatgg 180 agacgatcct tgtttgctac ctgatggtac tgatgcctatgctcttgcca aaaaggctgg 240 tcgatacaag cagattaaga gaaccgaggg agtgtcgacaacagacattg ttggacggat 300 gcttctttgt gttagagaga gatcatctga tgcacataaccactcgtcac tacaaaggca 360 gttcagtagt ggacatggtc agaaagttga tgatactggatctggaactg gaacaagagt 420 atctcatttt cttcccacat ctaggcgaat agttcaattctcaaatagca agggtcaggt 480 ccagattctc ggatagttta catagatggt gcttttgatctgttccatgc 530 2 157 PRT Zea mays 2 Ser Asp Asp Glu Ile Lys Ala Asn LysGly Pro Pro Val Thr Pro Leu 1 5 10 15 His Glu Arg Met Val Met Val ArgAla Val Lys Trp Val Asp Asp Ile 20 25 30 Ile Pro Asp Ala Pro Tyr Ala IleThr Glu Glu Phe Met Asn Lys Leu 35 40 45 Phe Asn Glu Tyr Asn Ile Asp TyrIle Ile His Gly Asp Asp Pro Cys 50 55 60 Leu Leu Pro Asp Gly Thr Asp AlaTyr Ala Leu Ala Lys Lys Ala Gly 65 70 75 80 Arg Tyr Lys Gln Ile Lys ArgThr Glu Gly Val Ser Thr Thr Asp Ile 85 90 95 Val Gly Arg Met Leu Leu CysVal Arg Glu Arg Ser Ser Asp Ala His 100 105 110 Asn His Ser Ser Leu GlnArg Gln Phe Ser Ser Gly His Gly Gln Lys 115 120 125 Val Asp Asp Thr GlySer Gly Thr Gly Thr Arg Val Ser His Phe Leu 130 135 140 Pro Thr Ser ArgArg Ile Val Gln Phe Ser Asn Ser Lys 145 150 155 3 544 DNA Oryza sativaunsure (205) n = A, C, G or T 3 tgtatcgtta gagagagatc agcttctgatagtcacaacc actcttcact acaaaggcag 60 ttcagtcacg ggcatggcca gaaaattgatgatagtggat ctggaagtgg aactaggata 120 tctcattttc ttcctacatc tcggagaatagttcagttct caaatagcag gggtccaggt 180 ccagattctc ggatagtgta catanatggtgcatttgatc tattccatgc tggacatgtt 240 gagatattgc gcctcgctcg agagcttgngagatttcctg cttgtgggta ttcacacaag 300 accagactat aagttcaaca agaggaccacatcgcccaat catgaacctc catnagagaa 360 gtttgagtgt tttggcttgc cgttatgttgatgaatgatc atggggctcc atgggatgtt 420 tccgaaagat atgatnanca catttaataattccnttggg tnttcatggg acanntgctg 480 ngaatatggn nttatgaagg atgattaatccatatgctgt tcaangggta tnggcatcta 540 ccgt 544 4 83 PRT Oryza sativaUNSURE (59) Xaa = any amino acid 4 Ser His Asn His Ser Ser Leu Gln ArgGln Phe Ser His Gly His Gly 1 5 10 15 Gln Lys Ile Asp Asp Ser Gly SerGly Ser Gly Thr Arg Ile Ser His 20 25 30 Phe Leu Pro Thr Ser Arg Arg IleVal Gln Phe Ser Asn Ser Arg Gly 35 40 45 Pro Gly Pro Asp Ser Arg Ile ValTyr Ile Xaa Gly Ala Phe Asp Leu 50 55 60 Phe His Ala Gly His Val Glu IleLeu Arg Leu Ala Arg Glu Leu Xaa 65 70 75 80 Arg Phe Pro 83 5 471 DNAGlycine max unsure (18) n = A, C, G or T 5 cttcgccaag ctcgtgcnctcggcgaccag ttgattgttg gggttgttag tgatgcagag 60 atcattgcca ataagggcccccccgttacc cctcttcacg aaaggttgat aatggtgaat 120 gcggtgaagt gggtnnatgaggttattcct gaagctccct atgcgataac cgaggagttc 180 atgaagaagc tttttgatgagtacaagata gattacatta ttcacgggga tgatccttgt 240 gttcttccgg atggaactgatgcttatgct catgctaaga aggctggtcg atataagcag 300 ataaagcgta ctgaaggggtttcacactga tattgttggt cgatcttctc tgtgtaaaga 360 aggtctatta ctgaaaaatcataatcattc ttctttacaa ancatcanca atggcatagt 420 cgagttgagc tgtcactgctgcactgnagt gactcgtata ntctttntgc n 471 6 108 PRT Glycine max UNSURE (46)Xaa = any amino acid 6 Leu Arg Gln Ala Arg Ala Leu Gly Asp Gln Leu IleVal Gly Val Val 1 5 10 15 Ser Asp Ala Glu Ile Ile Ala Asn Lys Gly ProPro Val Thr Pro Leu 20 25 30 His Glu Arg Leu Ile Met Val Asn Ala Val LysTrp Val Xaa Glu Val 35 40 45 Ile Pro Glu Ala Pro Tyr Ala Ile Thr Glu GluPhe Met Lys Lys Leu 50 55 60 Phe Asp Glu Tyr Lys Ile Asp Tyr Ile Ile HisGly Asp Asp Pro Cys 65 70 75 80 Val Leu Pro Asp Gly Thr Asp Ala Tyr AlaHis Ala Lys Lys Ala Gly 85 90 95 Arg Tyr Lys Gln Ile Lys Arg Thr Glu GlyVal Ser 100 105 7 493 DNA Triticum aestivum unsure (398)..(399) n = A,C, G or T 7 aacatggact atacagagga tgattcaaat ccatatgctg ctccaattgctatgggcatt 60 tatcataagc tggatagccc tttggacatc accactagta ctattataaggagaatagtt 120 tctaaccatg aagcctacca gaaacggaat gagaagaagg aagccagcgagaagaagtac 180 tatgacagta aaagtttgtc aatgggagag taagtgactt ctgaatagttctcctcaaga 240 agactgttcc tgggttcttt tggaggctct aacacaggtc acaaatggaaaccatcaagt 300 ggatcctcca attttaccgc tccattgtca tttttggcta tatacttaatgcttcaagat 360 gcatccttga tgcatgacag catgctgctg aattgggnnc cggacctggatgatttccaa 420 tgggcacttc aatgccacta ctacctaccc ccccggtana cnggttcnaaacgcnttacg 480 gccnatcttt tgg 493 8 57 PRT Triticum aestivum 8 Asp AspSer Asn Pro Tyr Ala Ala Pro Ile Ala Met Gly Ile Tyr His 1 5 10 15 LysLeu Asp Ser Pro Leu Asp Ile Thr Thr Ser Thr Ile Ile Arg Arg 20 25 30 IleVal Ser Asn His Glu Ala Tyr Gln Lys Arg Asn Glu Lys Lys Glu 35 40 45 AlaSer Glu Lys Lys Tyr Tyr Asp Ser 50 55 9 525 DNA Triticum aestivum unsure(485) n = A, C, G or T 9 tgaaaattgg cgttgttaag gtcaagtctg gcattatggaaggtccatct acaaatttga 60 tcgaatttat cgacttgagt cagaaaagtt ttgccattgcggctggatcg attttgttta 120 acccaacatt ctggaatatc gttgcgcgaa aagaatatcatgataagtcc ctaactaaac 180 ttgctggtgg caatgcacgc ctcggctgtt atatactcgcagttaccatc ttttgcttgg 240 ggatattccg agattttctc tatgagcgcg ccctccgtgatcagcctacc atgccactct 300 tgttgacaac cccctttcag cttctaagcc cttgttttagttatttcagg caatatccta 360 gtcatatcct caatgtgggc cctcggaata actggtacataccttggtga ttatttcgga 420 attctaatgg acgaaatggt gacgggcttc ccgtttaatgttaccgacgc accatgtatt 480 acggnagcac aatgagtttc ccaaggacag cactaatccttggaa 525 10 86 PRT Triticum aestivum 10 Thr Asn Leu Ile Glu Phe Ile AspLeu Ser Gln Lys Ser Phe Ala Ile 1 5 10 15 Ala Ala Gly Ser Ile Leu PheAsn Pro Thr Phe Trp Asn Ile Val Ala 20 25 30 Arg Lys Glu Tyr His Asp LysSer Leu Thr Lys Leu Ala Gly Gly Asn 35 40 45 Ala Arg Leu Gly Cys Tyr IleLeu Ala Val Thr Ile Phe Cys Leu Gly 50 55 60 Ile Phe Arg Asp Phe Leu TyrGlu Arg Ala Leu Arg Asp Gln Pro Thr 65 70 75 80 Met Pro Leu Leu Leu Thr85 11 1436 DNA Zea mays 11 ccgtaccttc cgaagctgca cgcctgcagg taccggtccggaattcccgg gtcgtatcca 60 cgcgttcggt tagtggtctc aagtgggtcg atgaggtcgttcccaatgca ccctatgaga 120 taacagaaga attcatgaat actctcttca acaagtacagcattgattac attattcatg 180 gagatgatcc ttgtcttcta cctgatggca ctgatgcatatgcgctagcg aagaaggtcg 240 ggcgttacaa gcaaatcaag cgaacagaag gtgtctcgagcactgacata gttgggagga 300 tattgctaac attcaggcag aaagatgctg acactgatttaagtgttgtc gttgctgaga 360 agtctggaga gaaatcaaat gatgaagtga aaagtcagctatctcatttc cttccaactt 420 ctcgccggat catgcagttt tcaaatgggc aggctccttcgccaggtgct cgtgttgtct 480 atgtagatgg cacatttgat cttttccacg ctggccatgttgagttcctc aggagtgcca 540 gacaacttgg tgactttctt cttgtgggta tctatgacgacgagtcgatc agggatagaa 600 gaggctgtcg tcctataatg catctccatg agcgtactcttggtgttctt gcctgccgtt 660 atgttgatga agtcattatt ggtgcaccat gggaagtttctaaggacatg atcactacgt 720 ttaacatttc attggttgtc catgggactg tagctgagggcaattcagct ggtgaaattg 780 atccttatgc tgttccaaag agcatgggga ttttccagacaatcaggagc ccaaaatcta 840 taacaacatt gtcagtggcg acaagaatag ttgacaatccatgaagctta caagaagagg 900 aacctgaaaa agaaggctag cgaagaccgg tactacacacaaaagaaatt cgtttctgga 960 gactagtgct gcacaaggag tgtatatttc tgccagcagtccgaggaaat gcatgtgcca 1020 ccctgtattg atgttattct acagcagaga ctggaacagataatcagcaa tagaaaggtc 1080 acaatgatag tttgagcaat ggtgtggatg gaccaagctagggaagagag agagagagag 1140 agagagagaa aactgtcaca ttcttctgct gtcgcccttttaggaacgct cacatgacat 1200 gaggagttca cataaacgat tctttttctt tctgactttgttatccccgt atggggattt 1260 atatatatct gtagctgaag gccttcagca aaccgttgttgtatacttgt gtgttgttac 1320 ttaacagcgt gtgtagtgat tagcactgca cactccctgattgtaccatg gtatattgaa 1380 tgtttatact gctggaagaa aaaaaggggg accttgtgatggtcagatgt ttaatt 1436 12 293 PRT Zea mays 12 Val Pro Ser Glu Ala AlaArg Leu Gln Val Pro Val Arg Asn Ser Arg 1 5 10 15 Val Val Ser Thr ArgSer Val Ser Gly Leu Lys Trp Val Asp Glu Val 20 25 30 Val Pro Asn Ala ProTyr Glu Ile Thr Glu Glu Phe Met Asn Thr Leu 35 40 45 Phe Asn Lys Tyr SerIle Asp Tyr Ile Ile His Gly Asp Asp Pro Cys 50 55 60 Leu Leu Pro Asp GlyThr Asp Ala Tyr Ala Leu Ala Lys Lys Val Gly 65 70 75 80 Arg Tyr Lys GlnIle Lys Arg Thr Glu Gly Val Ser Ser Thr Asp Ile 85 90 95 Val Gly Arg IleLeu Leu Thr Phe Arg Gln Lys Asp Ala Asp Thr Asp 100 105 110 Leu Ser ValVal Val Ala Glu Lys Ser Gly Glu Lys Ser Asn Asp Glu 115 120 125 Val LysSer Gln Leu Ser His Phe Leu Pro Thr Ser Arg Arg Ile Met 130 135 140 GlnPhe Ser Asn Gly Gln Ala Pro Ser Pro Gly Ala Arg Val Val Tyr 145 150 155160 Val Asp Gly Thr Phe Asp Leu Phe His Ala Gly His Val Glu Phe Leu 165170 175 Arg Ser Ala Arg Gln Leu Gly Asp Phe Leu Leu Val Gly Ile Tyr Asp180 185 190 Asp Glu Ser Ile Arg Asp Arg Arg Gly Cys Arg Pro Ile Met HisLeu 195 200 205 His Glu Arg Thr Leu Gly Val Leu Ala Cys Arg Tyr Val AspGlu Val 210 215 220 Ile Ile Gly Ala Pro Trp Glu Val Ser Lys Asp Met IleThr Thr Phe 225 230 235 240 Asn Ile Ser Leu Val Val His Gly Thr Val AlaGlu Gly Asn Ser Ala 245 250 255 Gly Glu Ile Asp Pro Tyr Ala Val Pro LysSer Met Gly Ile Phe Gln 260 265 270 Thr Ile Arg Ser Pro Lys Ser Ile ThrThr Leu Ser Val Ala Thr Arg 275 280 285 Ile Val Asp Asn Pro 290 13 1947DNA Oryza sativa unsure (490) n = A, C, G or T 13 gcaccagccc gcatctgccaccctgatctc ctcgcgtcgc gccctcccct cacctcgaat 60 ggcgatacct cctccacccacgcgccccaa tccctaaccc tagatccctc ctcccacgcc 120 ctctcgcgca cgaccaatctccctcttcct tcctgttgtg tctctctcgc actgtccaat 180 ctcccaattc attatggaggcgggggcggg gagcagcagc gccaagctgg tggcggcgtg 240 cgtcatcggc gggatcgtgctgggggcatc cgtggtcgcg ctccacctcg ccggccccgt 300 cgccattccc gccctgcgccgtcgacgcgc tccgccgcgg ttccgccgcg gacgacggcg 360 ccccgtgcgc gtctacatggatggctgctt cgacatgatg cactacggcc actgcaacgc 420 gctgcgccag gcgcgcgccctcggggacga gctcatcgtc ggcgttgtca gcgaccacga 480 gatcaccgcn aacaagggcccgccggtcac gcccctccac gagaggttga taatggtccg 540 tgctgtaaaa tgggtacacgatgttattcc agatgcacct tacgccataa ctgaggattt 600 catgaataaa ttattcaatgagtataatat agattatatc atccatggcg atgatccttg 660 tctgctccca gatggtactgatgcatatgc tcttgccaaa aaggttggtc gatttaaaca 720 gattaaaaga accgaaggagtgtcaacgac agacattgtt ggaagaatgc ttcttcgtgt 780 tagagagaga tcagcttctgatagtcacaa ccactcttca ctacaaaggc agttcagtca 840 cgggcatggc cagaaaattgatgatagtgg atctgaaagt ggaactagga tatctcattt 900 tcttcctaca tctcggagaatagttcagtt ctcaaatagc aggggtccag gtccagattc 960 tcggatagtg tacatagatggtgcatttga tctattccat gctggacatg ttgagatatt 1020 gcgcctcgct cgagagcttggagatttcct gcttgtgggt attcacacag accagactat 1080 aagttcaaca agaggaccacatcgcccaat catgaacctc catgagagaa gtttgagtgt 1140 tttggcttgc cgttatgttgatgaagtgat cattggtgct ccatgggatg tttcgaaaga 1200 tatgattacc acatttaatatttcgttggt tgttcatggg acaattgctg agaatatgga 1260 ctttatgaag gatgatttaaatccatatgc tgttccaagg gctatgggca tctaccgtag 1320 actggagagc cctttagacatcactactag tactatcata aggaggatag ttgctaacca 1380 tgaagcctac cagaaacggaacgagaagaa agaagccagt gagaagaagt actacgacag 1440 taaaagcttt gtcaatggagagtaacttag gaacaggtct tgcattaatg ctattgccca 1500 gaagtttagt tcacaaccttctggcacaaa tgcagcggtt agatgatcca caattttaca 1560 gtcttgtggt aactattctcatgttgctga tatagctcag gaaacttcag atgcaaccct 1620 gatgatggtg ctgacttgggtgatgctgga gaccctattt tcctgtatat ggggctttgt 1680 ggctgccaat accagctgtgttattttgag atgggtagtt tttttttttt tgtttttttg 1740 ttcaggattg ttgtagagtatgaatgttaa gcttgattaa ctattcctga tgctttattt 1800 cggagttgcc aggtatatgtggcatcatct tatgagagtc cttttctcat atattttggt 1860 acacttctgt tatgatctggaactgagcaa ctgatatttg tgtgggtcgg tgacagcaac 1920 tgtgtctgga atctggatgttttttcc 1947 14 423 PRT Oryza sativa 14 Met Glu Ala Gly Ala Gly Ser SerSer Ala Lys Leu Val Ala Ala Cys 1 5 10 15 Val Ile Gly Gly Ile Val LeuGly Ala Ser Val Val Ala Leu His Leu 20 25 30 Ala Gly Pro Val Ala Ile ProAla Leu Arg Arg Arg Arg Ala Pro Pro 35 40 45 Arg Phe Arg Arg Gly Arg ArgArg Pro Val Arg Val Tyr Met Asp Gly 50 55 60 Cys Phe Asp Met Met His TyrGly His Cys Asn Ala Leu Arg Gln Ala 65 70 75 80 Arg Ala Leu Gly Asp GluLeu Ile Val Gly Val Val Ser Asp His Glu 85 90 95 Ile Thr Ala Asn Lys GlyPro Pro Val Thr Pro Leu His Glu Arg Leu 100 105 110 Ile Met Val Arg AlaVal Lys Trp Val His Asp Val Ile Pro Asp Ala 115 120 125 Pro Tyr Ala IleThr Glu Asp Phe Met Asn Lys Leu Phe Asn Glu Tyr 130 135 140 Asn Ile AspTyr Ile Ile His Gly Asp Asp Pro Cys Leu Leu Pro Asp 145 150 155 160 GlyThr Asp Ala Tyr Ala Leu Ala Lys Lys Val Gly Arg Phe Lys Gln 165 170 175Ile Lys Arg Thr Glu Gly Val Ser Thr Thr Asp Ile Val Gly Arg Met 180 185190 Leu Leu Arg Val Arg Glu Arg Ser Ala Ser Asp Ser His Asn His Ser 195200 205 Ser Leu Gln Arg Gln Phe Ser His Gly His Gly Gln Lys Ile Asp Asp210 215 220 Ser Gly Ser Glu Ser Gly Thr Arg Ile Ser His Phe Leu Pro ThrSer 225 230 235 240 Arg Arg Ile Val Gln Phe Ser Asn Ser Arg Gly Pro GlyPro Asp Ser 245 250 255 Arg Ile Val Tyr Ile Asp Gly Ala Phe Asp Leu PheHis Ala Gly His 260 265 270 Val Glu Ile Leu Arg Leu Ala Arg Glu Leu GlyAsp Phe Leu Leu Val 275 280 285 Gly Ile His Thr Asp Gln Thr Ile Ser SerThr Arg Gly Pro His Arg 290 295 300 Pro Ile Met Asn Leu His Glu Arg SerLeu Ser Val Leu Ala Cys Arg 305 310 315 320 Tyr Val Asp Glu Val Ile IleGly Ala Pro Trp Asp Val Ser Lys Asp 325 330 335 Met Ile Thr Thr Phe AsnIle Ser Leu Val Val His Gly Thr Ile Ala 340 345 350 Glu Asn Met Asp PheMet Lys Asp Asp Leu Asn Pro Tyr Ala Val Pro 355 360 365 Arg Ala Met GlyIle Tyr Arg Arg Leu Glu Ser Pro Leu Asp Ile Thr 370 375 380 Thr Ser ThrIle Ile Arg Arg Ile Val Ala Asn His Glu Ala Tyr Gln 385 390 395 400 LysArg Asn Glu Lys Lys Glu Ala Ser Glu Lys Lys Tyr Tyr Asp Ser 405 410 415Lys Ser Phe Val Asn Gly Glu 420 15 1866 DNA Glycine max unsure (592) n =A, C, G or T 15 gcacgagcat tctacaattc gctacgcatt tcattccatt ccattccattttgtgtgggt 60 gctacgaatg aggaaggaga agaagcggtg ctagaataga tagatacatagatagagaag 120 agacatcatc gaacatctcg aaagagaggg aaaatgggta gttacgaggcgttgacggag 180 aagccggcga cggcgacgaa gtgggtggtg acgtgcatgg tgggaggggtgatcgtgggg 240 gtgtcactgc tgggtgcata ctcgagccag ctctggaaga gccgaagacgcaacaagaag 300 cccgttcgcg tctacatgga tggctgcttc gacatgatgc attatggccattgcaatgcc 360 cttcgccaag ctcgtgccct cggcgaccag ttgattgttg gggttgttagtgatgcagag 420 atcattgcca ataagggccc ccccgttacc cctcttcacg aaaggttgataatggtgaat 480 gcggtgaagt gggtggatga ggttattcct gaagctccct atgcgataaccgaggagttc 540 atgaagaagc tttttgatga gtacaagata gattacatta ttcacggggatngatccttg 600 tgttcttccg gantggaact gatgcttatg ctcatgctaa gaaggctggtcgantataag 660 cagataaagc gtactgaagg ggtttccagc actgatattg ttggtcgcatgcttctctgt 720 gtaagagaaa ggtctattac tgaaaaaaat cataatcatt cttctttacaaagacaattc 780 agcaatggcc atagtccgaa gtttgaagct ggtgcatctg ctgcaactgcaagtggaact 840 cgtatatctc attttttgcc tacatctcgt agaattgttc agttctcaaatgggaggggt 900 ccaggacctg attctcgcat tgtatatata gatggtgctt ttgatctctttcatgctgga 960 catgttgaga tcttgaggct tgctagggat cttggagatt ttcttcttgttggaatacac 1020 actgatcaga cagtcagtgc aactagagga tcgcatcgtc ctatcatgaatcttcatgaa 1080 agaagtctaa gtgttttagc atgtcgctat gtggatgagg ttataattggtgccccatgg 1140 gagatttcca aagatatgct cactacattt aacatctcat tagttgttcatggaaccatt 1200 gcagaaagta atgattttca gaaggaagaa tgcaatccat atgctgttcctattagcatg 1260 ggcatcttca aagttttaga aagtccttta gatataacta ctactacaataattagaagg 1320 attgtttcaa atcatgaggc ataccagaac cgaaataaga agaagggtgaaagtgagaaa 1380 agatactacg agggcaagag tcatgtgtct gaagaataat tcatgtctgtcgtttggagc 1440 acagacatag gaggatacca agctttttct cttttttgag aaatgagttttggttcaact 1500 tggtgcacaa agttggatat tgtgttcagt gcgtctacag gttatgatttgtcaaactta 1560 ttgaaccaac aacttaccta cagttgatca caagtatgaa agcgttccccaaataaattc 1620 gtgattaact aaattatctg ttaaatgagg tatacttgaa tagactcgcgaaccgaaatg 1680 ttcaacttat gctaccagcc gaacacaact cattttcttc attttttcttttcttttaaa 1740 tgacctatac tgtattattg ctgtgtgaga agttgttgaa gtattatgttgtctgattaa 1800 gtattgattt ttttgtttga taaactagtg aaaatatatc cgttaaatgactattgaata 1860 gttttt 1866 16 421 PRT Glycine max UNSURE (147) Xaa =any amino acid 16 Met Gly Ser Tyr Glu Ala Leu Thr Glu Lys Pro Ala ThrAla Thr Lys 1 5 10 15 Trp Val Val Thr Cys Met Val Gly Gly Val Ile ValGly Val Ser Leu 20 25 30 Leu Gly Ala Tyr Ser Ser Gln Leu Trp Lys Ser ArgArg Arg Asn Lys 35 40 45 Lys Pro Val Arg Val Tyr Met Asp Gly Cys Phe AspMet Met His Tyr 50 55 60 Gly His Cys Asn Ala Leu Arg Gln Ala Arg Ala LeuGly Asp Gln Leu 65 70 75 80 Ile Val Gly Val Val Ser Asp Ala Glu Ile IleAla Asn Lys Gly Pro 85 90 95 Pro Val Thr Pro Leu His Glu Arg Leu Ile MetVal Asn Ala Val Lys 100 105 110 Trp Val Asp Glu Val Ile Pro Glu Ala ProTyr Ala Ile Thr Glu Glu 115 120 125 Phe Met Lys Lys Leu Phe Asp Glu TyrLys Ile Asp Tyr Ile Ile His 130 135 140 Gly Asp Xaa Ser Leu Cys Ser SerGly Xaa Glu Leu Met Leu Met Leu 145 150 155 160 Met Leu Arg Arg Leu ValXaa Tyr Lys Gln Ile Lys Arg Thr Glu Gly 165 170 175 Val Ser Ser Thr AspIle Val Gly Arg Met Leu Leu Cys Val Arg Glu 180 185 190 Arg Ser Ile ThrGlu Lys Asn His Asn His Ser Ser Leu Gln Arg Gln 195 200 205 Phe Ser AsnGly His Ser Pro Lys Phe Glu Ala Gly Ala Ser Ala Ala 210 215 220 Thr AlaSer Gly Thr Arg Ile Ser His Phe Leu Pro Thr Ser Arg Arg 225 230 235 240Ile Val Gln Phe Ser Asn Gly Arg Gly Pro Gly Pro Asp Ser Arg Ile 245 250255 Val Tyr Ile Asp Gly Ala Phe Asp Leu Phe His Ala Gly His Val Glu 260265 270 Ile Leu Arg Leu Ala Arg Asp Leu Gly Asp Phe Leu Leu Val Gly Ile275 280 285 His Thr Asp Gln Thr Val Ser Ala Thr Arg Gly Ser His Arg ProIle 290 295 300 Met Asn Leu His Glu Arg Ser Leu Ser Val Leu Ala Cys ArgTyr Val 305 310 315 320 Asp Glu Val Ile Ile Gly Ala Pro Trp Glu Ile SerLys Asp Met Leu 325 330 335 Thr Thr Phe Asn Ile Ser Leu Val Val His GlyThr Ile Ala Glu Ser 340 345 350 Asn Asp Phe Gln Lys Glu Glu Cys Asn ProTyr Ala Val Pro Ile Ser 355 360 365 Met Gly Ile Phe Lys Val Leu Glu SerPro Leu Asp Ile Thr Thr Thr 370 375 380 Thr Ile Ile Arg Arg Ile Val SerAsn His Glu Ala Tyr Gln Asn Arg 385 390 395 400 Asn Lys Lys Lys Gly GluSer Glu Lys Arg Tyr Tyr Glu Gly Lys Ser 405 410 415 His Val Ser Glu Glu420 17 591 DNA Triticum aestivum 17 gcacgagaac atggactata cagaggatgattcaaatcca tatgctgctc caattgctat 60 gggcatttat cataagctgg atagccctttggacatcacc actagtacta ttataaggag 120 aatagtttct aaccatgaag cctaccagaaacggaatgag aagaaggaag ccagcgagaa 180 gaagtactat gacagtaaaa gctttgtcaatggagagtag tgacttctga atagatcttc 240 tcagaagact gttcctggat ctttgaggctctaacacagg tcacaaatga aaccatcaag 300 tgatcttcaa ttttaccgct ccattgtcatttttgctata tagcttagtg cttcagatgc 360 agtcttgatg catgacaggc agtgctgctgaattgggtgc ggaacctgga tgaatttcca 420 gtggccactt tcagtgccac tacctaccctatcctcccct gtataccggc ttccagagct 480 gctgttaccg gccgagttct ttttgagatctatagatctc tgagaattgt ggtacaagat 540 gaatgctaag gccagttaat tgtttgtttccctgttgcaa aaaaaaaaaa a 591 18 72 PRT Triticum aestivum 18 His Glu AsnMet Asp Tyr Thr Glu Asp Asp Ser Asn Pro Tyr Ala Ala 1 5 10 15 Pro IleAla Met Gly Ile Tyr His Lys Leu Asp Ser Pro Leu Asp Ile 20 25 30 Thr ThrSer Thr Ile Ile Arg Arg Ile Val Ser Asn His Glu Ala Tyr 35 40 45 Gln LysArg Asn Glu Lys Lys Glu Ala Ser Glu Lys Lys Tyr Tyr Asp 50 55 60 Ser LysSer Phe Val Asn Gly Glu 65 70 19 760 DNA Triticum aestivum 19 tgaaaattggcgttgttaag gtcaagtctg gcattatgga aggtccatct acaaatttga 60 tcgaatttatcgacttgagt cagaaaagtt ttgccattgc ggctggatcg attttgttta 120 acccaacattctggaatatc gttgcgcgaa aagaatatca tgataagtcc ctaactaaac 180 ttgctggtggcaatgcacgc ctcggctgtt atatactcgc agttaccatc ttttgcttgg 240 ggatattccgagattttctc tatgagcgcg ccctccgtga tcagcctacc atgccactct 300 tgttgacaaccccctttcag cttctagccc ttgttttagt tatttcaggc aatatcctag 360 tcatatcctcaatgtgggcc ctcggaataa ctggtacata ccttggtgat tatttcggaa 420 ttctaatggacgaaatggtg acgggcttcc cgtttaatgt taccgacgca cccatgtatt 480 acggaagcacaatgagtttc ctagggacag cactattctt tggaaagcct gctggaataa 540 tcttgacaacggaggtttta gttacctaca tgattgcatt gagactggaa aatcctttca 600 cagccaatatctatgccaag agaggtagca ttgccacgag gccttctcac gagaaagagc 660 tatagaaatttaaggttgaa taatttctca ttaaacctag ccaaaattag aatttcaatc 720 tataaattctagctcaataa aaaaaaaaaa aaaaaaaaaa 760 20 209 PRT Triticum aestivum 20 MetGlu Gly Pro Ser Thr Asn Leu Ile Glu Phe Ile Asp Leu Ser Gln 1 5 10 15Lys Ser Phe Ala Ile Ala Ala Gly Ser Ile Leu Phe Asn Pro Thr Phe 20 25 30Trp Asn Ile Val Ala Arg Lys Glu Tyr His Asp Lys Ser Leu Thr Lys 35 40 45Leu Ala Gly Gly Asn Ala Arg Leu Gly Cys Tyr Ile Leu Ala Val Thr 50 55 60Ile Phe Cys Leu Gly Ile Phe Arg Asp Phe Leu Tyr Glu Arg Ala Leu 65 70 7580 Arg Asp Gln Pro Thr Met Pro Leu Leu Leu Thr Thr Pro Phe Gln Leu 85 9095 Leu Ala Leu Val Leu Val Ile Ser Gly Asn Ile Leu Val Ile Ser Ser 100105 110 Met Trp Ala Leu Gly Ile Thr Gly Thr Tyr Leu Gly Asp Tyr Phe Gly115 120 125 Ile Leu Met Asp Glu Met Val Thr Gly Phe Pro Phe Asn Val ThrAsp 130 135 140 Ala Pro Met Tyr Tyr Gly Ser Thr Met Ser Phe Leu Gly ThrAla Leu 145 150 155 160 Phe Phe Gly Lys Pro Ala Gly Ile Ile Leu Thr ThrGlu Val Leu Val 165 170 175 Thr Tyr Met Ile Ala Leu Arg Leu Glu Asn ProPhe Thr Ala Asn Ile 180 185 190 Tyr Ala Lys Arg Gly Ser Ile Ala Thr ArgPro Ser His Glu Lys Glu 195 200 205 Leu 209

What is claimed is:
 1. An isolated polynucleotide comprising a firstnucleotide sequence selected from the group consisting of: (a) a firstnucleotide sequence encoding a polypeptide of at least 40 amino acidsthat has at least 80% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:2, 4, 6, 8, 10, 18, and 20; (b) a second nucleotide sequencecomprising a polypeptide of at least 200 amino acids that has at least80% identity based on the Clustal method of alignment when compared to apolypeptide selected from the group consisting of SEQ ID NOs:12, 14, and16; and (c) a third nucleotide sequence comprising a complement of thefirst or second nucleotide sequences.
 2. The isolated polynucleotide ofclaim 1, wherein the first nucleotide sequence comprises of a nucleicacid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5,7, 9, 11, 13, 15, 17, and
 19. 3. The isolated polynucleotide of claim 1wherein the nucleotide sequences are DNA.
 4. The isolated polynucleotideof claim 1 wherein the nucleotide sequences are RNA.
 5. A chimeric genecomprising the isolated polynucleotide of claim 1 operably linked to atleast one suitable regulatory sequence.
 6. An isolated host cellcomprising the chimeric gene of claim
 5. 7. A host cell comprising anisolated polynucleotide of claim
 1. 8. The host cell of claim 7 whereinthe host cell is selected from the group consisting of yeast, bacteria,and plant.
 9. A virus comprising the isolated polynucleotide of claim 1.10. A polypeptide selected from the group consisting of: (a) apolypeptide of at least 40 amino acids that has at least 80% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 18, and20; and (b) a polypeptide of at least 200 amino acids that has at least80% identity based on the Clustal method of alignment when compared to apolypeptide selected from the group consisting of SEQ ID NOs:12, 14, and16.
 11. A method of selecting an isolated polynucleotide that affectsthe level of expression of a phosphatidylcholine biosynthetic enzymepolypeptide in a plant cell, the method comprising the steps of: (a)constructing an isolated polynucleotide comprising a nucleotide sequenceof at least one of 30 contiguous nucleotides derived from an isolatedpolynucleotide of claim 1; (b) introducing the isolated polynucleotideinto a plant cell; (c) measuring the level of polypeptide in the plantcell containing the polynucleotide; and (d) comparing the level ofpolypeptide in the plant cell containing the isolated polynucleotidewith the level of polypeptide in a plant cell that does not contain theisolated polynucleotide.
 12. The method of claim 11 wherein the isolatedpolynucleotide consists of a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and
 19. 13. Amethod of selecting an isolated polynucleotide that affects the level ofexpression of a phosphatidylcholine biosynthetic enzyme polypeptide in aplant cell, the method comprising the steps of: (a) constructing anisolated polynucleotide of claim 1; (b) introducing the isolatedpolynucleotide into a plant cell; (c) measuring the level of potypeptidein the plant cell containing the polynucleotide; and (d) comparing thelevel of polypeptide in the plant cell containing the isolatedpolynucleotide with the level of polypeptide in a plant cell that doesnot contain the polynucleotide.
 14. A method of obtaining a nucleic acidfragment encoding a phosphatidylcholine biosynthetic enzyme polypeptidecomprising the steps of: (a) synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 30 contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and acomplement of such nucleotide sequences; and (b) amplifying a nucleicacid sequence using the oligonucleotide primer.
 15. A method ofobtaining a nucleic acid fragment encoding a phosphatidylcholinebiosynthetic enzyme polypeptide comprising the steps of: (a) probing acDNA or genomic library with an isolated polynucleotide comprising atleast one of 30 contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15, 17, and 19 and a complement of such nucleotide sequences;(b) identifying a DNA clone that hybridizes with the isolatedpolynucleotide; (c) isolating the identified DNA clone; and (d)sequencing a cDNA or genomic fragment that comprises the isolated DNAclone.
 16. A composition comprising the isolated polynucleotide ofclaim
 1. 17. A composition comprising the isolated polypeptide of claim10.
 18. An isolated polynucleotide of claim 1 comprising a nucleotidesequence having at least one of 30 contiguous nucleotides.
 19. A methodfor positive selection of a transformed cell comprising: (a)transforming a host cell with the chimeric gene of claim 5; and (b)growing the transformed host cell under conditions which allowexpression of a polynucleotide in an amount sufficient to complement anull mutant to provide a positive selection means.
 20. The method ofclaim 19 wherein the host cell is a plant.
 21. The method of claim 20wherein the plant cell is a monocot.
 22. The method of claim 20 whereinthe plant cell is a dicot.
 23. A method of altering the level ofexpression of a phosphatidylcholine biosynthetic enzyme in a host cellcomprising: (a) transforming a host cell with the chimeric gene of claim5; and (b) growing the transformed host cell produced in step (a) underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of altered levelsof a phosphatidylcholine biosynthetic enzyme in the transformed hostcell.
 24. A method for evaluating at least one compound for its abilityto inhibit the activity of a phosphatidylcholine biosynthetic enzymesthe method comprising the steps of: (a) transforming a host cell with achimeric gene comprising a nucleic acid fragment encoding aphosphatidylcholine biosynthetic enzyme, operably linked to at least onesuitable regulatory sequence; (b) growing the transformed host cellunder conditions that are suitable for expression of the chimeric genewherein expression of the chimeric gene results in production of thephosphatidylcholine biosynthetic enzyme encoded by the operably linkednucleic acid fragment in the transformed host cell; (c) optionallypurifying the phosphatidylcholine biosynthetic enzyme polypeptideexpressed by the transformed host cell; (d) treating thephosphatidylcholine biosynthetic enzyme polypeptide with a compound tobe tested; and (e) comparing the activity of the phosphatidylcholinebiosynthetic enzymes olypeptide that has been treated with a testcompound to the activity of an untreated phosphatidylcholinebiosynthetic enzyme polypeptide, and selecting compounds with potentialfor inhibitory activity.